A number of in vitro techniques have been employed to measure the increased bactericidal capacity of macrophages

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DESTRUCTION OF LISTERIA MONOCYTOGENES IN VITRO

George L. Spitalny Robert J. North

INTRODUCTION

An analysis of acquired cell-mediated immunity to bacterial infection ultimately requires an investigation of the bactericidal capacity of host macrophages in vitro. This stems from the fact that there is no way of knowing whether increased resistance in vivo depends on the generation of more macrophages or macrophages more efficient at destroying bacteria. For this reason, past investigations of acquired resistance to tuberculosis, brucellosis, salmonellosis, and listeriosis have involved, at some stage in the investigations, the demonstration that animals with acquired antibacterial resistance possess macrophages that express increased bactericidal activity in vitro (1). It is now known that cell-mediated antibacterial immunity, although specifically mediated by sensitized T cells, is nonspecifically expressed, in that macrophages of the immune host display an increased capacity

for inactivating a variety of unrelated bacteria. Listeria

MONONUCLEAR PHAGOCYTES 6 8 5 All rights of reproduction in any form reserved.

ISBN 0-12-044220-5

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monocytogenes is a useful organism for measuring this non- specific bactericidal activity.

A number of in vitro techniques have been employed to measure the increased bactericidal capacity of macrophages.

Many of these have employed macrophages adherent to glass or plastic, and many have measured bactericidal activity by de- termining changes against time in the number of cell-associated bacteria capable of forming colonies on nutrient agar.

A similar method is described here. It involves allowing peritoneal macrophages to adhere to and spread on glass cover-

slips, washing away all nonadherent cells, and challenging the resulting macrophage "monolayers" with an excess of Listeria.

Phagocytosis is allowed to proceed for a short period and the bacteria not associated with cells are then thoroughly washed away. Changes against time in the number of cell-associated colony-forming bacteria are then determined by plating serial dilutions of sonicates of macrophage monolayers on nutrient agar. In this way, the rate of bacterial kill can be determined (2) .

II. MATERIALS

A. Reagents 1. Animals

Mice are the most popular animals, although the procedure can be used to measure the bactericidal activity of macrophages from any animal, particularly since long-term macrophage cultures are not required.

2. Listeria monocytogenes

A strain must be chosen with at least moderate virulence for mice. The method for increasing virulence by repeated passage in mice is described by North and Spitalny, this volume, as is the method for obtaining a log phase culture of Listeria for macrophage challenge.

B. Dissecting Materials

Bell jar containing chloroform-soaked surgical gauze for killing mice

Two sterile 3-cm^ syringes fitted with 22-gauge needles (Pharmaseal Labs, Glendale, California, #7200)

50-ml Plastic tubes (Falcon Plastics, Oxnard, California,

#2070)

Ethanol, 70%

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Blunt end, 5 1/2 in. stainless scissors (Clay Adams, Parsippany, New Jersey

Cork board and pins

Hemocytometer for counting peritoneal cells (Scientific Products, McGraw Park, Illinois. #B3180

C. Tissue Culture Materials

A number of 35 x 10 mm tissue culture Petri dishes (Corn- ing Glass Works, Corning, New York, #25000, or Falcon Plastics, Oxnard, California, #3001)

A number of 12- or 13-mm diameter circular coverslips (Clay Adams, Parsippany, New Jersey, #3350), which are cleaned and sterilized by dipping in 70% ethanol and flaming

Small forceps for firmly holding coverslips

RPMI 1640 medium (GIBCO, Grand Island, New York, #430- 1800) containing 5% heat-inactivated (56°C for 1 hr) fetal bovine serum (RPMI-FBS) with or without 5 U/ml of preservative- free heparin (Sigma, St. Louis, Missouri, #3125)

Incubator at 37°C supplied with humidified air containing 5% CO?

D. Materials for Bacterial Challenge and Enumeration Petroff-Hauser chamber for enumerating Listeria

Sonicator set at 50% maximum output to break-up chains of bacteria and to free organisms phagocytosed by macrophages

(Biosonik, Brownwill Scientific, Rochester, New York

Bacterial colony counter (New Brunswick Scientific, Edison, New Jersey)

A number of 5-ml plastic tubes (Falcon Plastics, Oxnard, California, #2058) containing 0.9-ml physiological saline for serial dilutions

Bacterial culture plates (Falcon Plastics, Oxnard, Califor- nia, #1056) containing Trypticase-soy agar prepared as described by North and Spitalny, this volume.

Plastic 15-ml tubes containing 1 ml of 0.1% WR 1339 detergent (Ruger Chemical Co., Irvington, New Jersey) in sterile distilled water

A 2-liter aspirator bottle with outlet connected to a rubber tube fitted with adjustable clamp and hemostat. The bottle is filled with sterile saline containing 0.5% heat- inactivated fetal bovine serum.

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I I I . PROCEDURE

Mice are killed with chloroform vapor, their abdomens swabbed with 70% ethanol and the skin of their abdomens clipped carefully with scissors without perforating the peritoneal cavity. The clipped skin is held and pulled anterior- ally to expose the peritoneal lining. A 3-cm³ syringe with a 22-gauge needle is used to infuse 1.5 ml of heparinized RPMI- FBS into the peritoneal cavity, and the peritoneum massaged with the blunt end of a sterile pipette. The peritoneal fluid is then carefully withdrawn into a separate 3-cm³ syringe. In spite of the fact that only 1.5 ml of culture medium are in- fused, it is possible, with a little practice, to withdraw most of this volume into the syringe. The small volume of RPMI-FBS is used in order to obtain a relatively large number of cells per unit volume. This avoids the need to centrifuge and resuspend the cells, a procedure that results in cell clumping. The peritoneal cells of several mice are pooled, the number of cells per unit volume determined by counting a sample with a hemocytometer, the volume adjusted to give 5 x 106 cells/ml, and 1 ml of the suspension added to each of a number of 35 x 10 mm petri dishes containing three 13-mm diameter circular coverslips. Since the aim is to obtain

"macrophage monolayers" on the coverslips, an adequate number of cells per milliliter must be added to the petri dishes.

If the number of cells per milliliter is too low, the volume added can be increased.

The macrophages are allowed to settle, adhere to, and spread on the coverslips for at least 2 hr in an incubator at 37°C with 5% C02· The coverslips are removed with forceps and, while being held firmly with forceps, are subjected to the shear force of a jet of saline-FBS delivered from a Pasteur pipette and fed by gravity from an overhead aspirator bottle.

The rate of flow of fluid is controlled by an adjustable clamp on the connecting tubing. This washing procedure thoroughly removes all nonadherent cells. The coverslips are then placed

(cells-side up) into new 35 x 10 mm petri dishes containing 1 ml of RPMI-FBS and incubated an additional hour at 37°C be- fore bacterial challenge.

A sample of a log phase culture (1-2 x 10^/ml) of Listeria is appropriately diluted, counted with a Petroff-Hauser chamber, and the number of bacteria in the culture calculated.

The culture is then diluted to 5 x 10⁶ bacteria/ml in RPMI-FBS and subjected to ultrasound (50% maximum output) for 3 sec to break up chains of bacteria. The 1 ml of medium overlaying the macrophages on coverslips is aspirated and quickly replaced with 1 ml of medium containing the bacteria. The petri dishes

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are placed in the incubator, and the macrophages allowed to ingest bacteria for 20 min. The medium is then quickly removed and replaced with 5 ml of fresh medium to dilute the remaining uningested bacteria and greatly reduce any further phagocytosis. Each coverslip is then picked up with forceps and subjected to the same washing procedure as described above to remove nonadherent cells. This serves to remove all bacteria that are not cell-associated. The coverslips are then placed in new 35 x 10 mm petri dishes with 1 ml of fresh RPMI- FBS. Three coverslips from separate dishes are removed and dropped into a 15-ml plastic tube (cell side up) containing WR 1339 of 0.1% WRI1339 detergent in distilled water. The remaining plates are placed back in the incubator and bacterial inactivation is allowed to proceed. The three coverslips that were placed in the tubes represent triplicates of the T0 time point. Each of these coverslips are subjected to two 3-sec bursts of ultrasound (50% maximum output) to disrupt the cells and release intracellular bacteria. Immediately after sonica- tion, 0.1 ml of the sonicate is subjected to 10-fold serial dilution in 5-ml tubes containing 0.9 ml of saline, and each dilution is plated on TSA. If the foregoing procedure is ad- hered to, it is only necessary to plate the undiluted, 10-fold and 100-fold dilutions in order to obtain countable numbers of bacterial colonies 24 hr later. Three coverslips are removed

from the incubator at 15, 30, 60, 90, 120, and 180 min and subjected to the same procedure. These time points are desig- nated T^, T2, Γ3, etc.

IV. CALCULATION OF DATA

The numbers of bacterial colonies from known dilutions are recorded and the numbers of viable bacteria in the cell sonicates for each time point calculated in triplicate. The means are calculated and plotted against time. The data can be pre- sented as changes against time in the log^o number of viable bacteria per monolayer, or as changes in the percent bacteria killed against time. The latter is calculated according to the formula

Number of colony forming units T±, Τ2, Γ3 . . . Tn

Number of colony forming units Tn

The latter presentation is easiest to accept when two types of macrophages are being compared, because it is not essential that each type of macrophage be present on coverslips in equal number. This is because the assay assures that there is no competition for bacteria between macrophages in that the bacteria are fed uniformally to the monolayer by gravity. Conse-

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quently, each macrophage should ingest and destroy bacteria without being influenced by its neighbors. This means that within limits, the percent kill will be independent of the number of macrophages doing the killing. The washing procedure assures that practically all bacteria that are not cell-associated after 20 min are washed away. This avoids the need to use antibiotics to inhibit extracellular bacterial growth. This is just as well since there is evidence (3,4) that antibiotics en- ter macrophages and aid in intracellular killing.

V. CRITICAL COMMENTS

An inherent problem with in vitro bactericidal assays is that all bacteria are not completely ingested by macrophages at the same time. Even though the assay described here results in suitable numbers of bacteria becoming associated with macrophages firmly enough to avoid being washed off by a strong

jet of saline, some of the bacteria are already completely in- teriorized at the time of washing, while others are still in the process of being ingested. The rate of bacterial inactivation from TQ on will depend, therefore, on the rate at which the partly ingested organisms are completely ingested, assuming, of course, that bactericidal mechanisms depend on complete in- gestion. This problem becomes serious when the bactericidal activities of normal and activated macrophages are being compared, because activated macrophages possess an intrinsically enhanced capacity for ingesting bacteria at a faster rate.

There is no way of knowing, therefore, whether increased bactericidal activity displayed by activated macrophages is caused by the possession of more potent bactericidal mechanisms, or whether it is simply the result of the same bactericidal mechanisms acting on more rapidly ingested bacteria. One way of overcoming this problem is to employ subagglutinating quantities of opsonic antibodies to ensure that bacteria are ingested by control and activated macrophages at similar rates and to similar extents. Assuring that this is achieved involves a lot of trial and error and painstaking microscopic examination of the cultures.

Acknowledgment

Supported by Grants AI-10351 from the National Institute of Allergy and Infectious Diseases, CA-21360 from the National Cancer Institute and by RR05705 from the Biomédical Research Support Grant Program, Division of Research Resources, National Institutes of Health.

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REFERENCES

1. R. J. North. Cell-mediated immunity and the response to infection. In "Mechanisms of Cell-Mediated Immunity"

(R. T. McCluskey and S. Cohen, eds.), pp. 185-220.

Wiley, New York, 1973.

2. G. L. Spitalny. Suppression of macrophage bactericidal activity in ascites tumors. J. Reticuloendothel. Soc.

20:223-235, 1980.

3. P. Cole and J. Brostoff. Intracellular killing of

Listeria monocytogenes by activated macrophages (Mackaness system) is due to antibiotics. Nature (London) 256:515- 517, 1975.

4. P. D'Arcy Hart. Critical approach to the technique of assessment of antibacterial effects of activated mouse peritoneal macrophages. In "Activation of Macrophages"

(W. H. Wagner, H. Hahn, and R. Evans, eds.), pp. 131-317.

American Elsevier, New York, 1974.